Project Summary/Abstract While HIV-1 infection can be treated with antiretroviral therapy, current drug combinations are unable to clear the integrated proviral reservoir that is established within host cells early during the HIV-1 life cycle. The mechanisms that maintain this reservoir are not fully understood. Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) is a mammalian deoxynucleoside triphosphate (dNTP) hydrolase identified to restrict the replication of HIV-1 in non-dividing cells. In addition to its role as a viral restriction factor, SAMHD1 has been implicated in the regulation of proinflammatory pathways. Inherited, loss-of-function mutations in SAMHD1 are associated with Aicardi-Goutires syndrome (AGS), a severe autoimmune encephalopathy characterized by spontaneously enhanced type I interferon signaling. Our lab has identified that SAMHD1 interacts with components of the nuclear factor ?B (NF-?B) pathway and down-regulates its activity. We have also found that SAMHD1 suppresses HIV-1 gene expression and binds to the promoter of the virus. Because NF-?B is a major player in the transactivation of the HIV-1 promoter, the central hypothesis of this proposal is that SAMHD1 suppresses NF-?B activation and viral transcription to regulate the host antiviral innate response to HIV-1 infection. To test this hypothesis, the proposal addresses the following aims: Aim 1 will evaluate the functional link between SAMHD1-mediated suppression of NF-?B activation and HIV-1 gene expression. Aim 2 will determine whether SAMHD1 binds to the HIV-1 promoter in a sequence-specific manner by mapping the site(s) of interaction between the protein and proviral DNA. Since the dNTPase and retroviral restriction functions of SAMHD1 are dependent on specific catalytic residues and tetramerization of the protein, Aim 3 will define the domain of SAMHD1 required to modulate NF-?B and HIV-1 gene expression and the oligomeric state in which the protein binds to the HIV-1 promoter. The proposed studies will expand upon our findings of SAMHD1-mediated regulation of antiviral inflammatory responses and viral gene expression to improve our understanding of persistent HIV-1 infection and the cellular mechanisms that prevent uncontrolled inflammation.